Maintenance of acid/base and electrolyte homeostasis by the kidney and the secretion and reabsorption of ions in the gastrointestinal tract involve the concerted activity of Na/H exchangers (NHEs), Cl/HCO3 exchangers (AEs), H,K-ATPases, and other transporters. Although these transporters are known to operate in coupled systems, there are few examples of such systems that are well understood in terms of the contribution that each transporter makes to the overall process, and the ancillary mechanisms that come into play if the activity of a given transporter is perturbed. This information could be obtained more easily if animal models with defects in specific transporters were available. Thus, in aim 1 we will use embryonic stem (ES) cell/gene targeting technologies to develop mouse models in which the expression of individual transporters is ablated. We have begun experiments in which ES cells containing disruptions of the genes encoding NHE2, NHE3, NHE4, and the colon/renal H,K-ATPase are being prepared and used for blastocyst-mediated transgenesis to develop mouse lines carrying the disrupted genes. Using this approach we have obtained chimeric animals for the H,K-ATPase and NHEs and germline transmission of the mutant H,K-ATPase gene. A similar strategy will be used to prepare mice with an AE1 gene from which the mRNA encoding the kidney variant can no longer be transcribed, and mice with altered AE2 genes in which expression of either the AE2a or AE2b transcripts, but not both, has been ablated. The viability of mutant offspring will be assessed by analysis of genotype frequency, birthweight, survival and growth rate, embryonic development, and tissue histology. In aim 2 the effects of gene disruption on renal control of acid/base and potassium homeostasis will be studied by monitoring blood gases, pH, HCO3, Na, and K under normal, acidotic, alkalotic, and K depleted conditions. The existence of compensatory changes along the nephron will be assessed morphologically by electron microscopy and functionally by measuring HCO3 flux and transporter activities in isolated tubules. The effects of gene disruption on secretion, absorption, and mucosal protection in the gastrointestinal tract will be studied by analysis of morphological changes and histopathology using both light and electron microscopy, and by analysis of gastrointestinal secretion and ion transport. In aim 3 will clone and characterize the NHE5 Na/H exchanger and additional isoforms of the H,K-ATPase and AE families for which there is experimental evidence.